Mitochondrial dysfunction underlies the pathology of several diseases including Alzheimer's disease and diabetes. O-GlcNAcylation of mitochondrial proteins is increasingly being recognized as a regulatory mechanism contributing to these pathologies. O-linked N-acetyl-glucosamine glycosylation (O-GlcNAcylation) is an abundant, reversible and highly dynamic post-translational protein modification that regulates signal transduction, apoptosis, proteasome activity, transcription, translation and nuclear transport. Deregulated O-GlcNAcylation has been linked to diabetes-related complications, cancer progression, neurodegeneration and includes mitochondrial dysfunction. We have recently identified glycosylated isoforms of multiple essential mitochondrial proteins. The recent discovery of a specific mitochondrial isoform of OGT (mOGT) localized to the inner mitochondrial membrane raises the possibility that mitochondrial O-GlcNAcylation plays a role in oxidative phosphorylation, mitochondrial integrity, redox signaling, and cell survival pathways. While previous studies showed that stable or transient overexpression of mOGT results in apoptosis, the role of endogenous mOGT in mitochondrial protein O-GlcNAcylation and mitochondrial function remains largely unknown. This collaborative proposal will test the hypothesis that mitochondrial OGT activity is critical to mitochondrial structure and function. Unveiling novel physiological roles of mOGT will not only help us understand how O- GlcNAcylation regulates mitochondrial function but will help us to understand how dysregulated O-GlcNAcylation of key proteins contribute to disease pathogenesis of diabetes, cancer and neurodegenerative diseases.